| Summary: | Molecular hydrogen (H2) is seen as an ideal replacement for fossil fuels; however the current methods of its synthesis are unsustainable and environmentally damaging. Research is therefore needed to devise new, more suitable modes of H2 derivation. Biohydrogen – i.e. H2 derived from cellular metabolism – is a particularly promising future fuel, owing to it being a truly renewable means to generate H2 that would contribute to neither pollutant nor greenhouse gas emissions. The predominant enzymes responsible for microbial H2 evolution are called hydrogenases. The model organism Escherichia coli produces H2 during mixed acid fermentation by the action of it native hydrogenases. However, the natural level of H2 produced is too low to meet current industrial demand for H2 or any future uses as a fuel. The context of the work presented in this Thesis was therefore towards the augmentation of E. coli H2 yield. This was addressed through a number of specific aims that employed synthetic biology techniques. The first aim was to engineer E. coli to heterologously express genes that lead to the biosynthesis of a foreign NADH-consuming [NiFe]-hydrogenase, native to Cupriavidus necator. It was shown that this hydrogenase was assembled in E. coli and it exhibited activity both in vitro and in vivo. Serendipitously, it was found that the native E. coli maturase system, critical for biosynthesis of the [NiFe] cofactor, could assemble a functional enzyme. The converse was also found: that the C. necator maturase operons were able to complement E. coli mutants defective in hydrogenase biosynthesis. A second approach to augment E. coli H2 yield utilised in this Thesis was the generation of synthetic chimeric metalloenzymes that might allow for new substrates to be used. One such chimera, consisting of a fusion between E. coli Hyd-3 and a ferredoxin from Thermotoga maritima, was characterised and shown to exhibit in vivo H2 evolution when co-produced with a heterologous pyruvate: ferredoxin oxidoreductase. This allowed pyruvate to be employed as a new electron donor for H2 production. In addition, the further characterisation of a chimeric complex combining subunits of E. coli Hyd-2 and Salmonella enterica thiosulfate reductase was undertaken. This allowed H2 production to be driven by respiratory electron donors such as glycerol 3-phosphate. An additional aim of this work was to develop an intracellular H2 biosensor in E. coli, with a view to obtaining a screening method that could be used to scan mutant libraries for increased H2 production. The strategy was to utilise the H2-sensing regulatory [NiFe]-hydrogenase (RH) from C. necator in E. coli to control reporter gene transcription. Synthetic operons were designed for optimum expression of RH-encoding genes, and the heterologous biosynthesis of the RH apparatus was established. Various reporter gene constructs were also generated. However, the system was not found to be functional, and further experiments needed to address this are discussed.
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